Method and composition for regulating the activity of regulatory t cells

ABSTRACT

The present invention provides a composition and method of regulating the activity of regulatory T cells. The present invention relates to a composition containing an antigen recognized by CD4 + CD25 +  regulatory T cells or an expression vector encoding such an antigen and a method of controlling an immune response from a mammal by administrating the composition to the mammal. Furthermore, the present invention provides effective means for suppressing a rejection reaction and a graft-versus-host reaction in transplantation and for prevention and treatment of an autoimmune disease or an allergic disease. Furthermore, an immunosuppression condition can be removed by the administration of Interferon-γ or a combination of Interleukin 12 and Interleukin 18. In other words, those cytokine actions and the sensitization with an SEREX antigen are suitably combined to artificially manipulate regulatory T cells, allowing the cells to be applied to an autoimmune disease, reactions accompanied with organ transplantation, allergic reaction, control of tumor immunity, and the like.

INDUSTRIAL FIELD TO WHICH THE INVENTION BELONGS

The present invention relates to a method of regulating the activity ofregulatory T cells involved in regulation of immunity in the living body(in vivo), in particular a composition having activity for suchregulation.

PRIOR ART

Human beings have a biological defense system to remove exogenousmaterials and simultaneously have established self-tolerance. Suchimmune responses are induced and regulated by interactions amongB-lymphocytes, T-lymphocytes, antibodies, and antigen-presenting cells(APCs). First, exogenous antigens are subjected to processing with theAPCs and then bound to major histocompatibility complex (MHC) class-1and class-2 molecules, followed by being presented to helper T cells. Asthe helper T cells recognize the exogenous antigens bound to MHCs, the Tcells are activated, thereby secreting cytokines to assist thedifferentiation from B-cells stimulated by the antigens toantibody-producing cells and also to accelerate the differentiation ofkiller T cells. Cells presenting the antigen are eliminated by thesecreted antibodies and the activated killer T cells. Thus, cellular andhumoral reactions for removing the exogenous antigens progress. That is,the T cells play a central role and recognize antigens to be targeted,initiating an immune response. For example, in an antitumor immuneresponse, it has been known for a long time that both CD4⁺ T cells andCD8⁺ T cells play an extremely important role (L. Gross, Cancer Res. 3:326-333 (1943); L. Old et al., Ann. N. Y. Acad. Sci. 101: 80-106 (1962);R. J. North, Adv. Immunol. 35: 89-155 (1984); P. D. Greenberg, Adv.Immunol. 49: 281-355 (1991); D. M. Pardoll and S. L. Topalian, Curr.Opin. Immunol. 10: 588-594 (1998)). Even though CD8⁺ CTLs (cytotoxic Tcells) are major effector cells having ability to directly destroy tumorcells both in vivo and in vitro, they have been considered to beeffecter cells that represent unique immune responses such that they arestrict on the specificity of antigenic peptides bound to MHC class I,while natural killer T (NKT) cells are considered to be effector cellsfor intrinsic immune response with a moderate restriction of antigenicspecificity. (M. J. Smyth et al., J. Exp. Med. 191:661-668 (2000); M. J.Smyth&D. I. Godfray, Nature Immunol. 1: 459-460; M. J. Smyth et al.,Curr. Opin. Immunol. 14: 165-171 (2002); T. Kawano et al., Proc. natl.Acad. Sci. USA 95: 5690-5693 (1998)). On the other hand, CD4⁺ T cells donot directly destroy tumor cells but they may play a basic role ofcontrolling antitumor immune responses via various mechanisms (K. Hunget al., J. Exp. Med. 188: 2357-2368 (1998); F. Ossendorp et al., J. Exp.Med. 187: 693-702 (1998); D. M. Pardoll & S. L. Toplian, Curr. Opin.Immunol. 10: 588-594 (1998); R. F. Wang, Trends. Immunol. 5: 269-276(2001)). The CD4⁺ helper T cells that have recognized tumor antigenicpeptides bound to MHC class II molecules amplify the activation andproliferation of CTLs by interaction with antigen-presenting cells(APCs). In contrast, CD4⁺CD25⁺ regulatory T cells (Treg) have been shownto be effective in preventing antitumor immune responses and thedevelopment of various autoimmune diseases (E. M. Schvach, Annu. Rev.Immunol. 18: 423-449 (2000); M. G. Roncarolo & M. K. Levings, Curr.Opin. Immunol. 12: 676-683 (2000); J. Shimizuetal, J. Immunol. 163:5211-5218 (1999); S. Sakaguchi et al., Immunol. Rev. 182: 18-32 (2001)).It, however, is not clearly shown what will be autoantigenic peptidesrecognized by Treg and what will be the difference of Treg from orrelationship of Treg with helper T cells with respect to their antigenicrecognition and functions. (S. Sakaguchi, Nature Immunol. 2: 283-284(2001); K. J. Maloy & F. Powrie, Nature Immunol. 2: 816-822 (2001); E.M. Shevach, Nature Rev. Immunol. 6: 389-400 (2002)).

In the quantitative and qualitative amplification of CTLs, a possibilitythat the helper T cells implicated in antitumor immune responsesrecognize a wide range of various antigens has been suggested forsuccessive cellular interactions among helper T cells, CTLs, and APCs(J. P. Ridge et al., Nature 393: 474-478 (1998); S. R. M. Bennett etal., Nature 393: 478-480 (1998); S. P. Schoenberger et al., Nature 393:480-483 (1998); Z. Lu et al., J. Exp. Med. 191: 541-550 (2000)). Theinventor of the present invention has found out that an extremelyenhanced CTL activity and enhanced ability to cause a tumor rejectionreaction can be induced by application of the SEREX (serologicalidentification of antigen by recombinant cDNA expression cloning)antigen together with the CTL recognition antigen (H. Nishikawa et al.,Proc. Natl. Acad. Sci. USA 98: 14571-14576 (2001); WO 03/000894 A1).Consequently, the inventor of the present invention has reached theinvention with respect to vaccine for expressing tumor specific immunitywhere a composition is utilized as a polynucleotide vaccine, whichincludes expression vectors encoding a CD4⁺ helper antigen, preferably amolecule found by the SEREX method, and an antigen recognized by CD8⁺CTL, preferably a tumor antigen (WO 03/000894 A1), respectively.

The CD4⁺CD25⁺T regulatory cells are always produced in a functionallymatured state from the normal thymus. However, those cells are typicallyin an anergic state, so that they will strongly suppress the activationand proliferation of other T cells with the introduction of T cellreceptor (TCR) stimuli. On that occasion, it has been revealed thatstimulus transduction through CTLA-4 (cytotoxic T lymphocyte-associatedantigen 4) and GITR (glucocorticoid-induced tumor necrosis factorreceptor family) molecules which are constitutively expressed isrequired for expression of suppression ability (T. Takahashi et al., J.Exp. Med. 192: 303-310 (2000); S. Read et al., J. Exp. Med. 192: 295-302(2000); J. Shimizu et al., Nature Immunol. 3: 135-142 (2002)). Treg hasbeen considered to be composed of cells having a wide range ofspecificity to recognize autoantigen bound to MHC class II. However, theantigens are still not identified and there is no knowledge at all aboutmechanism of enhancing the immunosuppression with Treg.

DISCLOSURE OF THE INVENTION

Normal individuals such as mice and human beings possess their immunesystems as biological defense systems against exogenous materials, sothat they will not attack themselves. Such self-tolerance may be basedon three mechanisms of eliminating autoreactive immune cells,inactivating autoreactive immune cells, and suppressing immunity withTreg. Then, autoimmune disease may occur when the self-tolerance fails,while the self-tolerance allows cancer cells to proliferate. Inaddition, for organ or tissue transplantation, a mechanism to eliminateexogenous antigens makes the transplantation difficult. Now that itbecomes clear that Treg has an important role in immunosuppression in animmune system, it has been strongly expected to provide a method ofregulating the activity of Treg and to develop a pharmaceutical agentagainst diseases due to immune disorder, especially autoimmune diseases,transplantation rejection reactions, graft-versus-host reactions,allergic diseases, and so on.

The inventor of the present invention has reached the present inventionfrom his observation that the growth and metastasis of a tumor tissuehave been more accelerated when an expression vector encoding SEREXantigen was administered alone instead of administering it together withan expression vector encoding tumor antigen to a cancer-bearing mouse toevaluate antitumor effects thereof.

The present invention provides a composition containing an antigenrecognized by a CD4⁺CD25⁺ regulatory T cell. The present invention alsoprovides a method of suppressing an immune response of a mammal,including administering the composition to the mammal. The presentinvention further provides a method of preventing and/or treating anautoimmune disease, including administering the composition to a mammalin a pharmaceutically effective dosage. Alternatively, the presentinvention provides a use of the composition, in which the composition isused in production of a preventive agent and/or a therapeutic agent foran autoimmune disease.

The present invention further provides a method of preventing and/ortreating an allergic disease, including administering the composition toa mammal in a pharmaceutically effective dosage, or a use of thecomposition, in which the composition is used in production of apreventive agent and/or a therapeutic agent for an allergic disease.

The present invention also provides a method of suppressing a rejectionreaction and/or a graft-versus-host reaction in an organ or tissuetransplantation, including administering the composition to a mammal ina pharmaceutically effective dosage, or a use of the composition, inwhich the composition is used in production of a preventive agent and/ora therapeutic agent for suppressing a rejection reaction and/or agraft-versus-host reaction in an organ or tissue transplantation.

More specifically, there is provided a method of regulating the activityof regulatory T cells, where CD4⁺CD25⁺T cells (regulatory T cells) aresensitized by molecules (SEREX antigen) found by the SEREX (serologicalidentification of antigen by recombinant cDNA expression cloning) methodto enhance the activity of the regulatory T cells, while the activatedregulatory T cells are reduced by the use of Interferon-γ or shared useof Interleukin 12 and Interleukin 18. In particular, a composition thatenhances the activity of regulatory T cells may be a polynucleotidecontaining an expression vector encoding SEREX antigen. Thepolynucleotide may be administered alone into cells or may be fixed oncarrier particles or the like and then administered into cells.

An object of the present invention is to realize the suppression ofrejection and graft-versus-host reactions in transplantation, and thesuppression of autoimmune diseases and allergic reactions byartificially sensitizing regulatory T cells with antigen to enhanceimmunosuppression activity, and to provide a therapeutic methodeffective for patients having those diseases or pathologic conditions.

The present invention relates to a method of immunosuppression on thebasis of the finding that an immune reaction can be suppressed byadministering an expression vector encoding antigen recognized byCD4⁺CD25⁺T cells. Therefore, the present invention relates to acomposition containing an antigen recognized by CD4⁺CD25⁺T cells or anexpression vector encoding such an antigen. More specifically, theantigen recognized by the CD4⁺CD25⁺T cells is preferably a moleculefound by the SEREX method. In addition, the antigen may be a heteroantigen or an autoantigen, more preferably an autoantigen. The SEREXmethod is one developed in recent years by M. Pfreundschuh et al (Proc.Natl. Acad. Sci. USA94: 1914-1918 (1997)) and involves screening cDNAexpression libraries of human tumors using human sera. In the Internet,more than 1,800 different types of genes identified by the SEREX methodhave been registered as SEREX database (www.licr.org/SEREX.html). Theyinclude, but not limited to, heat shock proteins DnaJ-like2 (GenBankAccession Nos.:NM_(—)005494, XM_(—)028966, XM_(—)172161, XM_(—)052862,XM_(—)062754, XM_(—)093388, NM_(—)016306, NM_(—)012328, andNM_(—)005880), Galectin-8 (GenBank Accession Nos. :AH008815, AF193806,and AF193805), Poly(A)-binding protein (GenBank Accession No.:XM_(—)067844), and Ligase 1 (GenBank Accession No.: NM_(—)000234). Theterm “SEREX-identified molecules” as used herein refers to thosemolecules identified by the SEREX method.

In the present invention, polynucleotide that encodes an antigen may beDNA or RNA but not particularly limited as far as it can generate adesired immune response by administering the polynucleotide into a hostanimal according to the method of the present invention. Thepolynucleotide that encodes the antigen of the present invention may beany of those the nucleotide sequence of which is prepared byartificially deleting, substituting, inserting, and adding one or moreamino acid sequences by means of well-known procedures such as amutagenic method specific to a sequence site as far as a polypeptideencoded by the polynucleotide generates a desired immune response in thehost (see, edit. Ausubel et al., Current Protocols in Molecular Biology(1987) John Wiley & Sons, Section 8.1-8.5). In addition, as far as adesired immune response is generated in a host animal, a variant foundin the nature can be also isolated using a known hybridizationtechnology (see, edit. Ausbel et al., Current Protocols in MolecularBiology (1987) John Wiley & Sons, Section 6.1-6.4) and a geneamplification technology (PCR) (see, edit. Ausbel et al., CurrentProtocols in Molecular Biology (1987) John Wiley & Sons, Section6.1-6.4).

Furthermore, when a gene that encodes an antigen protein is known, aperson skilled in the art will carry out without difficulty analysis ofhydrophobic/hydrophilic regions on the amino acid sequence of such aprotein (Kyte and Doolittle, J. Mol. Biol. 157: 105-122 (1982)),analysis of a secondary structure (ChouandFasman, Ann. Rev. Biochem. 47:251-276 (1978)), estimation of an antigenic region in the amino acidsequence (see, for example, Anal/Biochem. 151: 540-546 (1985)), andsynthesis of a peptide corresponding to the estimated amino acidsequence to determine the antigenecity thereof according to the PEPSCANmethod (Nature 314 (1985) and JP 60-500684 A) or the like. Therefore, apolynucleotide that encodes a peptide fragment containing an epitopeportion defined by the above method can be produced by any of proceduresincluding a chemical synthesis and can be also used as an expressionvector for the antigen of the present invention.

The expression vector used in the present invention is a recombinantvector with the insertion of an antigen gene recognized by CD4⁺CD25⁺regulatory T cells. Examples of the vector to which an antigen gene isinserted include plasmids, phages, cosmids, viruses, and other vectorsconventionally used in the art. Any person skilled in the art willconstruct various kinds of plasmids and vectors using technologiesdescribed in, for example, Sambrook et al., (Ed.), Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.) andAusubel et al. (Ed.), Current Protocols in Molecular Biology (1987) JohnWiley & Sons.

Persons skilled in the art may suitably select factors for control ofexpression of antigen genes in host cells, such as promoters andterminators, from known control sequences appropriately and arrange themon the upstream or downstream of the genes on the basis of the types ofthe host cells and purposes, respectively. Therefore, the controlsequence used may be originated from the antigen or a heterologous one.If required, a marker such as an antibiotic resistance marker may beused in the expression vector of the present invention. Although manycommercially available vectors can be used, preferably those from whichpolynucleotide sequences unessential for the present invention areremoved may be used.

The composition of the present invention may be used as a naked plasmid.That is, the composition of the present invention may be used in theform of being packed in a ribosome or of one of various kinds of virusvectors including a retrovirus vector, an adenovirus vector, a vacciniavirus vector, a pox virus vector, an adenovirus-associated vector, andan HVJ vector (see, e.g., K. Adolph Virus Genome Method”, CRC Press,Florida (1996)), or may be used in the form of being coated on beads(carriers) such as colloidal gold particles. The composition of thepresent invention preferably has, but not limited to, a configurationsuch that a vector for the expression of antigen recognized by CD4⁺CD25⁺regulatory T cells is attached on carrier particles such as goldparticles to be introduced into the living body by means of a gene gunor the like. The technologies for coating carrier particles withpolynucleotides are known in the art (see, e.g., WO 93/17706). Finally,the polynucleotide can be prepared in a solution of saline or the likesuitable for administration to the living body. Other additional agentssuch as calcium ions may be used together to promote the intracellularincorporation of plasmid. Furthermore, if required, otherpharmaceutically acceptable agents that promote the transfection may beused together.

The composition of the present invention can be administered by any ofadministration methods. Preferably, however, the composition of thepresent invention is administered by means of injection, infusion, orgas-inductive particle-bombardment method (by an electron gun or thelike) through a suitable parenteral route such as an intravenous,intraabdominal, subcutaneous, or intracutaneous route, an intra-adiposetissue or intra-breast tissue route, or an inhalation or intramuscularroute; collunarium or the like through a mucosal route; or the like. Ofthose administration methods, genetic transformation techniques withaccelerated particles are described in U.S. Pat. Nos. 4,945,050 and5,240,842 and devices based on the modified methods thereof have beencommercially available (Biorad Laboratories).

The host animal species used in the present invention are not limited.However, specific examples of the species include mammals includingmice, rats, rabbits, dogs, cats, pigs, sheep, caws, horses, and primatessuch as monkeys and human beings. Of those, for the host animals, humanbeings are more preferable.

DETAILED DESCRIPTION OF THE INVENTION

The antigen or expression vector used in the present invention may begenerally administered at an amount of 0.001 μg to 1 g, preferably 0.01μg to 10 mg, more preferably 0.1 μg to 100 μg per dosage even though thedosage may vary depending on the type or degree of disease, thesexuality, age, and body weight of the animal, an administrating route,and so on, and is not limited.

The immunosuppression state realized as a result of carrying out theabove process can be removed by administration of Interferon-γ oradministration of Interleukin 12 together with Interleukin 18. In otherwords, an appropriate combination of the action of cytokine and thesensitization with SEREX antigen allows regulatory T cells to beartificially modified and applied to an auto immune disease, a reactionwith organ transplantation, allergic reaction, and control of tumorimmunity.

Alternatively, furthermore, removal of an immunosuppression state may becarried out by the treatment with anti-CD4 antibody or anti-CD25antibody.

Therefore, the present invention provides a method and composition forcontrolling the activity of regulatory T cells.

Since the present invention has found that SEREX antigen promotes theimmunosuppression activity of regulatory T cells, there is shown amethod of controlling the activity of regulatory T cells for the firsttime.

Conventionally, even though various procedures and agents have beendeveloped for the treatments of autoimmune diseases and allergy as wellas the treatments for suppressing transplantation rejection andgraft-versus-host reaction, the effectiveness thereof reaches a limit.Therefore, those having higher effectiveness have been needed.

The present invention provides a method and composition extremelyeffective in controlling the action of CD4⁺CD25⁺ regulatory T cells theimportance of which in the living body has been clarified more and morein late years, bringing a breakthrough in the treatments with therapy ofautoimmune diseases and allergic diseases and organ and tissuetransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the immunization with SEREX antigen alonepromotes metastases to lung.

FIG. 2 is a graph showing that cells responsible for promotingmetastases to lung induced by the immunization with SEREX antigen aloneare CD4⁺ or CD4⁺CD25⁺ cells but not CD8⁺ cells.

FIG. 3 is a graph showing that transplantation of CD4⁺CD25⁺ T cells fromthe mouse immunized by SEREX antigen alone promotes metastases to lung.

FIG. 4 is a graph showing that the pre-treatment with SEREX antigenalone reduces the number of CD8⁺ T cells specific to mERK2 antigen.

In the figure, 9m-pulsed P1HTR denotes mERK2 9m antigen protein andp63-71(T)-pulsed P1HTR denotes the control antigen protein.

FIG. 5 is a graph showing in vivo preventive and therapeutic effects ofimmunization with mERK2 or 147HER2 and Dna J-like-2 on metastases tolung.

FIGS. 6, 7, 8, and 9 show results of measuring the diameters of tumorson the backs of the mice in Example 4.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to examples thereof. However, these examples have no intentionof restricting the scope of the present invention in any sense.

Reference Example Inhibition of Metastases to Lung by SimultaneousSensitization with SEREX Antigen and Tumor Antigen

In vivo injection of 3-methylcholanthrene-induced sarcoma CMS5m cellsoriginated from BALB/c into animals causes metastases to lung, so thatthe animals will be dead within 5 to 6 weeks. Thus, a model ofmetastases to lung was established by administrating 1×10⁶ CMS5m tumorcells of 0.1 ml in total into a BALB/C mouse through the lateral caudalvein thereof. mERK2 which is a mutated kinase was used as a tumorantigen, and DnaJ-like2 was used as an SEREX antigen. Immunization wasinitiated every other weak such that the immunization was initiatedbefore 14 days or 7 days from the tumor administration with 1) a plasmidencoding mERK2, 2) a mixture of the mERK2 plasmid and a plasmid encodinga control vector, and 3) a mixture of the mERK2 plasmid and a DnaJ-like2plasmid, or on the day of the tumor administration (on the same date),or after 5 days from the tumor administration (according to the methoddescribed in H. Nishikawa et al., Proc. Natl. Acad. Sci. USA 98:14571-14576 (2001)). After 28 days from the tumor administration, themice were sacrificed and the number of pulmonary nodules was countedunder dissecting microscopy. For each group, five animals were used. Theresults are shown by means±SEM thereof.

Before 14 days from the tumor administration, mice were immunized byplasmid encoding mERK2, resulting in complete prevention of metastasesto lung. Such a preventive effect could not be observed when theimmunization was carried out before 7 days from the tumor administrationand also after the tumor administration (see FIG. 5). In contrast, theimmunization with a combination of mERK2 plasmid and DnaJ-like2 plasmidprevented metastases to lung completely even after up to 5 days from thetumor administration (see FIG. 5). The presence or absence of metastaseswas confirmed by means of a histopathological examination. The numeralvalue in the figure shows means±SEM of five mice for each group.

Example 1 Promotion of Metastases to Lung by Sensitization with SEREXAntigen Alone

In the same model of metastases to lung as that of Reference Example,mice immunized by the plasmids encoding SEREX antigen or the controlvectors after 5 days from the tumor administration were sacrificed after28 days from the tumor administration, respectively. Then, the number ofpulmonary nodules was counted for each animal. When the animals wereimmunized by the respective plasmids of four different antigens (each ofthem was a mouse's protein), i.e., a heat shock protein DnaJ-like2, DNAligase 1, Galectin-8, and poly (A)-binding protein cytoplasmic, as theSEREX antigen, the number of the nodules was 130 to 170. In the case ofno immunization like that, the number of the nodules was 30 to 50. Onthe other hand, when the animals were respectively immunized by plasmidsencoding three mouse's molecules which could not be detected even afterrepeated SEREX analysis, i.e., glucose regulated protein, secretednexin, and TCP-1 zeta subunit (Cctz-1)-containing chaperon, nometastasis promotion was observed (FIGS. 1 a and 1 b). In addition, evenif the animals were respectively immunized by plasmids encoding threehuman SEREX antigens (Homo sapiens HMBA-inducible protein, humanretinoic acid response protein, and Homo sapiens hepatitis deltaantigen-responsive protein A), and different species of proteins such asovalbumin, no promotion of metastasis was observed (FIG. 1 a). In otherwords, it was suggested that the sensitization with the SEREX antigenalone as autoantigen promoted metastases.

Therefore, cell phenotypes involved in the metastatic promotion wereanalyzed. When the mouse was pre-treated with anti-CD4 monoclonalantibody or anti-CD25 monoclonal antibody, no metastatic promotion dueto the sensitization with DnaJ-like2 was observed at all (FIGS. 2 a and2 b). When the mouse was pre-treated with anti-CD25 monoclonal antibody,the number of metastatic nodules considerably decreased, compared withone which was not pre-treated (FIG. 2 b). In addition, when the mousewas pre-treated with anti-CD8 monoclonal antibody and sensitized withDnaJ-like2, the metastatic promotion was observed as much as oneobserved in the case of no antibody pre-treatment (FIG. 2 c). Thisresult showed that CD4⁺ or CD4⁺CD25⁺ T cells were responsible for themetastatic promotion.

Example 2 CD4⁺CD25⁺ T cells Sensitized by SEREX Antigen PromoteMetastases to Lung

For directly validating the results obtained in Example 1, CD4⁺CD25⁺ Tcells sensitized by SEREX antigen was transplanted in a tumor-inoculatedmouse and the presence or absence of metastatic promotion were observed.That is, when CD4⁺CD25⁺ T cells were transplanted from a mouse immunizedby DnaJ-like2 antigen into a tumor mouse, metastases to lung wasconsiderably promoted. In contrast, when CD4⁺CD25⁻ T cells weretransplanted, nearly no promotion of metastases to lung was observed. Inaddition, significant metastatic promotion was observed whenunsensitized CD4⁺CD25⁺ T cells were transplanted, compared with notransplantation. In this case, however, the degree of metastaticpromotion was extremely small, compared with the case in whichsensitized CD4⁺CD25⁺ T cells were transplanted (FIG. 3).

Example 3 Pre-Treatment with SEREX Antigen Suppresses the Activity ofCD4⁺ T cells but not CD8⁺ T Cells

The inventor et al. of the present invention have previously reportedthat the administration of each of plasmids encoding tumor antigen andSEREX antigen to the tumor-inoculated mouse could induce a strongantitumor immune response, while considerably enhancing the helperfunction of CD4⁺ T cells, and besides the amplification of CD8⁺ T cellresponse was depended on CD4⁺ T cells (H. Nishikawa et al., Proc. Natl.Acad. Sci. USA 98: 14571-14576 (2001); WO 03/000894 A1).

In view of the above, it was studied whether or not the immune responsesof CD8⁺ T cells and CD4⁺ T cells changed with respect to the suppressionon an antitumor immune response confirmed in Example 2. A 9-mer ofmutated kinase (i.e., a peptide mERK2 9m) was used as a tumor, antigen.A mouse was pre-immunized with DnaJ-like2 and then immunized with aplasmid encoding mERK2 9m alone or together with a plasmid encodingDnaJ-like2, followed by analyzing mERK2 9m specific CD8⁺T cells by theELISPOT assay. If mERK2 9m was used alone, the pre-treatment withDnaJ-like2 did not affect CD8⁺ T cells (FIG. 4 a). However, in the caseof combined immunization, enhanced helper activity observed at the timeof no pre-treatment with DnaJ-like2 was completely disappeared by thepre-treatment with DnaJ-like2 (FIG. 4 b). Consequently, it was foundthat the immunization with SEREX antigen suppressed the helper activityof CD4⁺T cells but not CD8⁺T cells. The analysis with α-GalCer-CD1dtetramer confirmed that the number of local natural killer T (NKT) cellsdecreased.

From Example 1, Example 2, and Example 3 described above, as an exampleof the antitumor immune response of a mouse tumor metastasis model, itis experimentally proved that the treatment with a plasmid encoding theSEREX antigen alone suppresses the entire immune response by suppressingthe activity of CD4⁺ helper T cells as a result of the activation of theCD4⁺CD25⁺ regulatory T cells.

Example 4 Control of Alloimmune Response by Control of Activity ofRegulatory T Cells

The inventor of the present invention has identified a wild-typeautoantigen without mutation recognized by the immune system of acancer-bearing individual by means of the SEREX method using acancer-bearing mouse serum (SEREX-identified autoantigen) TheSEREX-identified autoantigen may be recognized by CD4 cells after beingrecognized by IgG. An enhanced helper effect can be exerted when thoseSEREX-identified autoantigen and cytotoxic T cell (CTL) recognizingcancer rejection antigen are used for co-immunization using aHelios-type gene gun (Gene GunR, Helios, Co., Ltd.).

On the other hand, for the immunization with SEREX-identifiedautoantigen alone, it is found that the tumor in a lung metastasis modelof a murine sarcoid becomes worse and the immunity is regulatedsuppressively.

This mechanism is considered to be used for the immunization of soleSEREX-identified autoantigen with a decrease in NKT by activatingregulatory CD4⁺CD25⁺T cells. In this example, for investigating how animmunosuppression effect due to the immunization with soleSEREX-identified autoantigen participates in an alloimmune response intransplantation or the like, the tumor originated from a mouse ofanother strain was inoculated into a mouse immunized by theSEREX-identified autoantigen alone, and the effect of the inoculation onproliferation was investigated.

(1) Materials and Methods

Mice: BALB/c(H-2d) and C57BL/6(H-2b) mice were used.

Plasmids: SEREX-identified autoantigen genes (DnaJ-like2, Mus DNA Ligase1 (Ligase 1), Mus poly(A)-binding protein, and cytoplasmic 1 (Poly (A))were incorporated into the respective pBK-CMV plasmids and then coatedon gold particles.

Gene Introduction (Immunization): Immunization with the SEREX-identifiedautoantigen alone was carried out by intracutaneously supplying into theabdomen of a mouse by using the Helios-type gene gun.

Tumor Inoculation: After one week from the second immunization with theSEREX-identified autoantigen alone, a BALB/c mouse was inoculated with amelanoma cell line B16 originated from C57BL/6 and a C57BL/6 mouse wasinoculated with a sarcoma cell line CMS5a originated from BALB/c, whichwere subcutaneously inoculated in the backs thereof at a concentrationof 1×10⁶.

After tumor inoculation, the diameter of the tumor of the mouse's backwas measured over time (See FIGS. 6 to 9).

Antibodies: An anti-CD25 antibody was administered at a concentration of0.25 mg after concentration and fractionation of the antibody withammonium sulfate from hybridoma PC61.

As the anti-GITR (Glucocorticoid-induced TNF receptor superfamily 18)antibody, a hybridomaDTA-1 (Prof. Shimon Sakaguchi, Kyoto Univ.)peritoneal fluid was diluted 8 times and then 200 μl (25 μl) thereof wasprovided. As the anti-CD4 antibody, a hybridoma GK1.5 peritoneal fluidwas diluted 8 times and then 200 μl (25 μl) thereof was provided. Forthe respective antibodies, the administration of antibody was performedonce immediately before the inoculation of tumor.

(2) Results and Discussion

For BALB/c mice, rejection of the tumor originated from C57BL/6 mice wasprolonged by immunization of SEREX-identified autoantigen and some ofthe mice were dead of tumor.

This phenomenon was cancelled by the administration of anti-CD25antibody or the administration of anti-GITR antibody. Thus, theCD4⁺CD25⁺ T cells may participate.

As a result of suppressive regulation of the immune response by theimmunization of SEREX-identified autoantigen, an alloimmune response mayhave also been reduced.

1.-19. (canceled)
 20. A method of immunosuppressing a mammal, comprisingadministering a composition comprising an antigen recognized by aCD4⁺CD25⁺ regulatory T cell to the mammal.
 21. The method as claimed inclaim 20, in which the mammal is a human being.
 22. The method asclaimed in claim 20, additionally comprising a step of administeringInterferon-γ to boost the immune response from the mammal.
 23. Themethod as claimed in claim 20, additionally comprising a step ofadministering Interleukin 12 and Interleukin 18 to boost the immuneresponse from the mammal.
 24. The method as claimed in claim 20, inwhich the immunosuppressing is used to prevent and/or treat anautoimmune disease.
 25. The method as claimed in claim 20, in which theimmunosuppressing is used to prevent and/or treat an allergic disease.26. The method as claimed in claim 20, in which the immunosuppressingsuppresses a rejection reaction and/or a graft-versus-host reaction inan organ or tissue transplantation.
 27. The method as claimed in claim20, in which the antigen recognized by the CD4⁺CD25⁺ regulatory T cellcomprises a molecule identified by a SEREX method.
 28. The method asclaimed in claim 20, in which the composition comprises an expressionvector that encodes an antigen recognized by a CD4⁺CD25⁺ regulatory Tcell.
 29. The method as claimed in claim 28, in which the antigenrecognized by the CD4⁺CD25⁺ regulatory T cell comprises a moleculeidentified by the SEREX method.
 30. The method as claimed in claim 27,in which the molecule identified by the SEREX method comprises anautoantigen.
 31. The method as claimed in claim 27, in which themolecule identified by the SEREX method comprises one selected from thegroup consisting of DnaJ-like2, Galectin-8, poly(A)-binding protein, andLigase-1.
 32. The method as claimed in claim 28, in which a gene gun isused for administration of the composition.
 33. The method as claimed inclaim 21, additionally comprising a step of administering Interleukin 12and Interleukin 18 to boost the immune response from the human being.34. The method as claimed in claim 21, in which the immunosuppressing isused to prevent and/or treat an autoimmune disease.
 35. The method asclaimed in claim 21, in which the immunosuppressing is used to preventand/or treat an allergic disease.
 36. The method as claimed in claim 21,in which the immunosuppressing suppresses a rejection reaction and/or agraft-versus-host reaction in an organ or tissue transplantation. 37.The method as claimed in claim 29, in which the molecule identified bythe SEREX method comprises an autoantigen.
 38. The method as claimed inclaim 29, in which the molecule identified by the SEREX method comprisesone selected from the group consisting of DnaJ-like2, Galectin-8,poly(A)-binding protein and Ligase-1.
 39. The method as claimed in claim29, in which a gene gun is used for administration of the composition.